The invention relates to acquisition methods for fragment ion spectra of peptides in RF ion trap mass spectrometers, which are usually coupled to separation methods such as chromatography or capillary electrophoresis.
Current mass spectrometric research into biopolymers such as peptides, proteins and genetic material is frequently coupled with fast separation methods, such as liquid chromatography (LC) or capillary electrophoresis (CE). The objective here is often to fragment the biopolymer ions in the mass spectrometer in order to obtain information about the sequences of the biopolymer building blocks and about modifications of these building blocks. For peptides and proteins this means information about the sequence of the amino acids and further information about phosphorylation, glycosylation and other changes to the original protein structure as determined by a gene. It is therefore necessary to obtain fragment ion spectra with high information content. The types of mass spectrometer for this objective have become known as “tandem mass spectrometers”. The methods of acquiring fragment ion spectra with tandem mass spectrometers are often abbreviated to MS/MS or MS2.
Tandem mass spectrometers comprise a first mass spectrometer to select ions of a certain type, a fragmentation device, in which these selected ions are fragmented, and a second mass spectrometer to analyze the fragment ions. In ion trap mass spectrometers, these processes of selecting, fragmenting and analyzing the fragment ions can also be performed in temporal succession within the same ion trap; this is then termed “tandem-in-time”, in contrast to “tandem-in-space” in the case of spatially separated mass spectrometers.
In proteomics, it is frequently necessary to analyze thousands of peptides which have been obtained from an enzymatic digest of a complex protein mixture and separated by either liquid chromatography or electrophoresis. Qualitatively good fragment ion spectra contain information concerning the sequence of the amino acids but, unfortunately, only relatively few qualitatively good fragment ion spectra are measured with the automatic acquisition technique. This is the problem addressed by the invention described below, particularly in the light of these very complex peptide mixtures. The invention relates particularly to the use of RF ion trap mass spectrometers according to Wolfgang Paul which, on the one hand, are particularly suited to this objective but, on the other, also have characteristic drawbacks compared to other types of tandem mass spectrometer.
A Paul ion trap generally consists of a ring electrode and two end cap electrodes. An RF voltage at the ring electrode generates a quadrupole RF alternating field in the interior, which drives ions back into the center regardless of their polarity. Without collision gas, the ions oscillate in the ion trap in this so called pseudopotential well. The frequency of these so called “secular” oscillations is strongly characteristic for the charge related mass m/z of the ions. However, the ion trap is normally filled with a collision gas, usually helium, at a pressure of some 10−2 Pascal, so that the oscillation is damped in a few milliseconds by a large number of gentle collisions and the ions arrive in relative calm in the center of the ion trap, forming a small cloud. The energetic states in the interior of the molecules are also reduced; this is termed “cooling” by the collision gas. The diameter of the ion cloud in the center of the ion trap is determined by the equilibrium between the centripetal force of the RF field and the centrifugal force of the Coulomb repulsion between the ions. The ions can be excited to swinging secular oscillations by a dipolar excitation alternating voltage across both end cap electrodes, particularly when the excitation frequency matches the secular oscillation frequency. This is termed “resonant excitation”.
The ions can be selectively ejected from the ion trap according to their mass by several known methods and can thus be measured in an ion detector as a mass spectrum. To acquire a fragment ion spectrum, all ion species of an ion source are first stored; the ion species which are not to be analyzed are then ejected using known methods so that only the ion species to be analyzed as “parent ions” remains in the ion trap. This process is termed “isolation” of the selected parent ions. These parent ions can now be fragmented, for example by forced collisions with the collision gas under continuous resonant excitation. The fragments which remain behind as ions can then be selectively ejected according to their mass and measured as a fragment ion spectrum. The fragment ion spectrum is also termed “daughter ion spectrum”.
The filling of the ion trap with ions for subsequent isolation of the parent ions must be controlled so that sufficient numbers of ions are still available for scanning the daughter ion spectrum. One such method of control is described in the publication of the patent application DE 197 09 086 A1 (corresponding to Patents GB 2 322 961 B, U.S. Pat. No. 5,936,241 A), for example.
Besides this type of ion trap, which is usually called a “three-dimensional ion trap”, there is also a “two-dimensional” or “linear” ion trap, which comprises four pole rods with end electrodes resembling apertured diaphragms. The manner of operation of this linear ion trap will not be discussed here. It must be incorporated into the basic idea of the invention, however, since the idea is not dependent on the type of ion trap, as long as this ion trap has quadrupole RF alternating fields and means for collisionally induced fragmentation.
For the stated aim of elucidating the structure of peptides, ion trap mass spectrometers are usually equipped with electrospray ion sources, which supply not only singly charged ions of the digest peptides but also doubly and triply charged ions, which are particularly suitable for fragmentation with a high information content. The conventional mode of fragmentation here is collisionally induced fragmentation (CID=collision-induced dissociation), in which the ions are forced to oscillate by means of resonant excitation within the ion trap; the ions collide with the collision gas molecules contained in the ion trap (usually helium, more rarely nitrogen), thereby absorbing energy before finally decomposing. Modern ion trap mass spectrometers are also equipped with fragmentation devices which are based on a transfer of electrons and produce a different fragmentation pattern. This fragmentation can be brought about in different ways, which are summarized here under the collective name “electron induced fragmentation” (EID=electron-induced dissociation). This fragmentation results either from the capture of low energy electrons (ECD=electron capture dissociation), from a transfer of the electrons from negatively charged ions to the positively charged analyte ions (ETD=electron transfer dissociation), or from the transfer of electrons from highly excited neutral particles (MAID=metastable atom-induced dissociation).
The two fundamentally different fragmentation methods, CID and EID, contain complementary information, and so are preferably applied to the same ion species, preferably even to ions of different charge states of this ion species.
A characteristic feature of collisionally induced fragmentation CID is that longer or heavier modifying side chains, for example phosphorylation, sulfate or glycosylation groups, are preferably split off from the chain of the amino acids as neutral fragments because, generally, they are bound with low binding energy. The fragment ion spectrum hence reflects only the naked chain of the amino acids, not their modifications. The knowledge concerning the modification is lost completely if their splitting off does not leave behind changes to the amino acids themselves. This is the case in rare cases only, such as the creation of dehydroxyserine when serine is dephosphorylated.
In the chain of the amino acids it is the peptide bonds which split during collisionally induced fragmentation CID, i.e. the bonds of the nitrogen atoms to carbon on the N-terminal side of the nitrogen. The ions thus created are termed b fragment ions if the N-terminal fragment remains as an ion charged with a proton, otherwise as a y fragment ion for the C-terminal fragment ion. If one starts with doubly charged ions, then it is frequently the case that both ions of the complementary b and y fragment ion pair occur.
In contrast, electron-induced fragmentation splits the bonds of the nitrogen atoms in the chain of the amino acids on the C-terminal side. The ions created are termed c ions or z ions. A cleavage rearrangement means that the fragmentation acts at the point where the proton which was neutralized by the electron had been attached. The fragmentation is extremely gentle; all modifications remain intact. It is favorable here to start with triply charged parent ions. The comparison of this EID fragment ion spectrum with a CID spectrum immediately shows which of the ions in the CID spectrum are of the b type and which are of the y type, since there are always fixed mass separations of 17 atomic mass units between the b ions of the CID spectrum and the c ions of the EID spectrum. Complementary to this, the y ions are always 16 atomic mass units heavier than the z ions. In addition, unusual masses for the mass separations between the ion signals in the EID spectrum immediately make it apparent which of the amino acids carries the modification and what mass this modification has. It is thus favorable to measure both the CID and the EID fragment ion spectrum for each peptide. If the time available does not allow this, then at least the EID fragment ion spectra for the modified peptide ions should be measured. Modified peptide ions can often be recognized by losses of neutral fragments of a specific mass, for example the dephosphorylation by the mass m=98 atomic mass units.
The upstream separation method for the biopolymers provides the mass spectrometer with the analyte substance, in this specific case a digest peptide, for only a few seconds. For the complex mixtures described above, several digest peptides are often supplied simultaneously at any one time; not infrequently even between ten and twenty digest peptides simultaneously. An ion trap mass spectrometer can acquire around three to five mass spectra per second, so the measurements must be carried out sparingly. The control programs of this ion trap mass spectrometer contain methods to automatically acquire fragment mass spectra; they are briefly described here:
Before a fragment ion spectrum will be measured, a continuous series of normal mass spectra are acquired. The normal mass spectra are stored digitally in the memory of the mass spectrometer. For each mass spectrum, an evaluation program is then used to determine in real time whether one or more digest peptides are in fact supplied in sufficient concentration. If this is the case, a mathematical analysis of the mass spectrum is then used to select which ion species is most favorable for the acquisition of a fragment ion spectrum. Analyses of this type are familiar to those skilled in the art; in particular, it is known how singly, doubly and triply charged ion species can be identified using the mass separations in the isotope pattern. Doubly or triply charged ions are best suited to collisionally induced fragmentation, so the most intensive ion species which occurs with a double or triple charge within a predetermined mass range, not listed in an exclusion table, is generally used for the acquisition of the next fragment ion spectrum. The exclusion table contains the mass values of those peptides which have already been analyzed in previous measuring cycles or which were marked as not of interest at the outset. The selected species of parent ion is then isolated in the ion trap and fragmented by resonant excitation in the next acquisition cycle; the fragment ions are then measured in the form of a fragment ion spectrum.
If a device for electron-induced fragmentation is present, the acquisition of an EID fragment ion spectrum most favorably begins with triply or four times charged parent ions. If time allows, it is advisable to immediately measure both the CID as well as the EID fragment ion spectra for all the ion species which occur.
For both modes of fragmentation there are method parameters which are generally set blindly by the automatic control software in the way that has, on average, proven favorable for ions of a digest peptide of this mass. This method has proven reasonably successful for peptides without modifications, but it is precisely for modified peptides that this method seems not to be sufficient. In a single liquid chromatographic separation run with automatic mass spectrometric analysis lasting several hours, a few thousand daughter ion spectra may be obtained with sufficiently high quality for a successful evaluation, which might, on the face of it, be considered a good result. However, since this run involves the acquisition of a total of 10,000 to 100,000 fragment ion spectra, the number of qualitatively good daughter ion spectra is much too low. Analyses show that the proportion of fragment ion spectra with adequate quality is frequently not more than ten percent and very rarely over 20 percent of the total number of fragment ion spectra acquired. The analytical objective of detecting all the analyte substances, if possible, has not been satisfactorily achieved as yet, a fact which, unfortunately, is all too frequently only established when these daughter ion spectra are used for an identity and structure search with the aid of “search engines” in protein sequence databases.
If one analyzes the collisionally induced fragmentation spectra more closely, it is possible to ascertain that, in particular, the modified peptides frequently do not provide good fragment spectra. In many cases, a modification group splits off from the peptide as a neutral fragment; the residual peptide is then no longer resonantly excited, but is quickly cooled in the collision gas; it can no longer decompose further under these conditions. The fragment spectrum then essentially comprises only one single dominant ion species, which still carries the same number of charges as the parent ions, but has less mass.
Peptide ions which are complexed with alkali ions are also distinguished by the occurrence of a dominant ion species in the fragment ion spectrum, but the dominant ion species carries one charge less than the selected parent ions. The alkali ion is lost here.
The spectra of electron-induced fragmentation also often exhibit only one single dominant peak, generally a radical ion which does not independently decay any further, but carries a lower charge than the parent ions.
A rough rule of thumb is that around five to fifteen percent of all fragment ion spectra exhibit such a dominant ion signal.